Muir, Anna P, Biek, Roman, and Mable, Barbara K (2014) Behavioural and
physiological adaptations to low-temperature environments in the common
frog, Rana temporaria. BMC Evolutionary Biology, 14 (1). p. 110. ISSN
1471-2148
Copyright © 2014 The Authors
http://eprints.gla.ac.uk/94746/
Deposited on: 27 June 2014
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Muir et al. BMC Evolutionary Biology 2014, 14:110
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RESEARCH ARTICLE
Open Access
Behavioural and physiological adaptations to
low-temperature environments in the common
frog, Rana temporaria
Anna P Muir*, Roman Biek and Barbara K Mable
Abstract
Background: Extreme environments can impose strong ecological and evolutionary pressures at a local level.
Ectotherms are particularly sensitive to low-temperature environments, which can result in a reduced activity period,
slowed physiological processes and increased exposure to sub-zero temperatures. The aim of this study was to
assess the behavioural and physiological responses that facilitate survival in low-temperature environments. In
particular, we asked: 1) do high-altitude common frog (Rana temporaria) adults extend the time available for larval
growth by breeding at lower temperatures than low-altitude individuals?; and 2) do tadpoles sampled from
high-altitude sites differ physiologically from those from low-altitude sites, in terms of routine metabolic rate (RMR)
and freeze tolerance? Breeding date was assessed as the first day of spawn observation and local temperature
recorded for five, paired high- and low-altitude R. temporaria breeding sites in Scotland. Spawn was collected and
tadpoles raised in a common laboratory environment, where RMR was measured as oxygen consumed using a
closed respiratory tube system. Freeze tolerance was measured as survival following slow cooling to the point when
all container water had frozen.
Results: We found that breeding did not occur below 5°C at any site and there was no significant relationship
between breeding temperature and altitude, leading to a delay in spawning of five days for every 100 m increase
in altitude. The relationship between altitude and RMR varied by mountain but was lower for individuals sampled
from high- than low-altitude sites within the three mountains with the highest high-altitude sites (≥900 m). In
contrast, individuals sampled from low-altitudes survived freezing significantly better than those from high-altitudes,
across all mountains.
Conclusions: Our results suggest that adults at high-altitude do not show behavioural adaptations in terms of
breeding at lower temperatures. However, tadpoles appear to have the potential to adapt physiologically to
surviving at high-altitude via reduced RMR but without an increase in freeze tolerance. Therefore, survival at
high-altitude may be facilitated by physiological mechanisms that permit faster growth rates, allowing completion
of larval development within a shorter time period, alleviating the need for adaptations that extend the time
available for larval growth.
Keywords: Routine metabolic rate, Freeze tolerance, Spawning temperature, Altitude, Scotland
* Correspondence: [email protected]
Institute of Biodiversity, Animal Health and Comparative Medicine, University
of Glasgow, Glasgow G12 8QQ, UK
© 2014 Muir et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
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Background
Stressful environments (environments outside the optimum
conditions for a particular species) can impose strong
ecological and evolutionary pressures at a local level [1,2].
Population persistence depends on the ability of individuals to respond to environmental stress through adaptive,
plastic or behavioural mechanisms that maximise fitness
[3]. Extremes of pH (common frog; [4]), water availability
(wild mustard [5]), and temperature (redband trout; [2])
have been observed to drive adaptive population divergence. High-latitudes and altitudes experience low temperatures that can result in shorter activity periods and
longer periods of freezing [3,6,7]. Plastic and adaptive
responses to low temperature environments have been
widely recorded (for a review see [8]) and can result in
cryptic divergence between populations inhabiting different temperature regimes (counter-gradient variation;
[9]). Temperature is often the major abiotic factor that
influences physiological mechanisms in ectotherms [10,11]
and growth slows in response to cold environments [12].
Reduced activity periods in low-temperature environments,
in combination with low-temperature driven growth-rate
reductions, can result in lower sizes at important lifehistory events such as metamorphosis and reproduction
[13]. Smaller sizes can translate to lower fitness when
weight is positively correlated with survival or reproductive success [13,14].
Assessing the mechanisms that facilitate survival in
challenging environments is important for understanding
how populations respond to ecological and evolutionary
pressures, particularly in a globally changing climate [15].
Potential responses to maximise size at important life
history stages in low-temperature environments include
altering metabolic rate (e.g. to allow more resources to
be allocated to growth; [6,16]), developmental period (e.g.
delaying sexual maturity; [17-19]), or temperature activity
range (e.g. breeding at lower temperatures; [20]). Populations that inhabit high-altitude environments experience
lower temperatures and shorter activity periods than their
low-altitude neighbours and offer an excellent opportunity
to assess how survival is facilitated in environments where
growth is constrained [21]. Amphibians are a particularly
good model for studying physiological and behavioural responses to growth constraints, as size at metamorphosis
is positively correlated with survival in the subsequent
terrestrial life-history stages [22,23].
Variation in metabolic rates between individuals is a
common occurrence in nature [24], but the effects on fitness are still relatively unknown [25]. Resting metabolic
rate, here defined as the energetic cost of self-maintenance
[26], has been linked to multiple physiological and behavioural traits including predator avoidance, foraging behaviour, swimming performance and growth [13,24]. Growth
imposes a significant physiological cost and can result in a
Page 2 of 11
trade-off with other physiological mechanisms [13,27,28],
especially when resources are limited [29]. As the vast majority of energy expenditure in ectotherms is maintenance
costs (80-85%), small differences in resting metabolic
rate can result in large differences in energy available
for growth [29]. An increased growth rate can result in
a larger size at important life history events and has
been linked to a reduced resting metabolic rate in sagebrush lizards at high-altitude [6], Sydney rock oysters
from growth rate-selected stock [30] and snapping turtles [31]. Attempts to assess the physiological trade-offs
facilitating higher growth rates in larval common frog
(Rana temporaria) at high latitudes have found no link
to reduced metabolic rates [16]. However, as temperature
is not linearly related to latitude in Sweden [7], where
these experiments were conducted, these results may
mask the true nature of the temperature-metabolic rate
relationship. Therefore, further research in a system with a
linear temperature change is required to elucidate the relationship between resting metabolic rate, growth rate and
temperature.
Another potential response to maximise size at important life-history events, is to increase the time available for growth prior to metamorphosis or reproduction
by extending development over multiple growth periods
[18]. The concept of delayed development, or diapause,
has been commonly observed in insects, often in terms of
cohort splitting where different cohorts within a population complete development at different times of the year,
or even in different years [18]. In amphibians, the period
immediately prior to metamorphic climax is accompanied
by a loss of weight [32], but a lower weight decreases the
chances of adult survival [22]. Therefore, overwintering
at a higher weight, but still at the larval stage, and metamorphosing the following year has the potential to increase survival, and has been recorded in a number of
temperate amphibian species [19,32]. However, low winter
temperatures at high-altitude can lead to prolonged periods of freezing [33]. Therefore, in order to survive, overwintering larval amphibians must be able to respond to
freezing temperatures via freeze avoidance (i.e. inhabiting
environments that buffer individuals from freezing temperatures) or freeze tolerance (survival of extensive
freezing of body fluids; [3,34,35]). Freeze tolerance depends on the ability to restrict ice formation to extracellular areas, which is mediated by accumulation of
low molecular weight carbohydrates in the blood [3,34].
The ability to tolerate freezing has been linked to glucose
accumulation in the blood, via release of liver glycogen, in
the frogs R. sylvatica, R. lessonae and R. esculenta [3,34],
and with glycerol accumulation in Hyla versicolor [36].
However, all previous studies have focussed on freeze
tolerance in adult amphibians and the potential for
freeze survival in the larval stage has, to the best of our
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knowledge, never been studied. The ability of a tadpole
to survive freezing would extend the time available for
growth to, and thus size at, metamorphosis in amphibians
breeding in temperate climates.
A third alternative response to larval growth constraints
would be for adults to adapt behaviourally rather than
amphibian larvae adapting physiologically. Adults have
the potential to expand the growing season for larvae by
breeding earlier in the year [20]. In temperate amphibians, breeding is closely linked to temperature [37] and
frequently occurs immediately after winter dormancy
(e.g. Bufo bufo, R. chinensis, R. sylvatica and R. temporaria;
[38,39]). By adults becoming active and breeding at lower
temperatures, larvae would have longer to grow and develop prior to winter dormancy. The longer time available
for growth would allow larvae to reach a larger size at
metamorphosis and thus have an increased chance of
survival as adults [22].
The common frog (R. temporaria) is the most widespread amphibian in Europe and occurs from zero to
2742 metres above sea level within its range, and to over
a thousand metres on the mountains of Scotland [40-42].
It is an explosive breeder, with communal spawning taking place immediately after winter dormancy [43]; a 5°C
temperature threshold is generally considered to initiate
activity and spawning [44]. R. temporaria larvae show
increased growth rates in response to low temperatures
experienced at high-latitudes and altitudes throughout
its range [16,45]. We have previously shown that local
adaptation to high-altitude environments occurs even in
the face of high gene flow, suggesting that temperature
exerts a strong selective pressure [45]. However, there
are also reports of R. temporaria overwintering as tadpoles
in Scotland, although it is currently unclear whether
this response is particularly linked to low-temperature
environments [19]. The mountains of Scotland offer an
excellent opportunity to study the responses that facilitate survival in low-temperature environments, as
there is continuous habitat along altitudinal gradients,
with temperature decreasing linearly by 0.65°C for every
100 m gain in altitude [40,46,47]. Individuals from highaltitude sites in Scotland experience substantially lower
temperatures than their low-altitude counterparts, with
an average mean annual temperature reduction of 4.5°C
at high- compared to low-altitude breeding sites [40].
The overall aim of this study was to assess the physiological and behavioural responses of common frogs in
Scotland that facilitate survival in low-temperature environments. In particular, this study answers the questions:
1) do high-altitude adults extend the time available for
larval growth by breeding at lower temperatures than
low-altitude individuals?; and 2) do tadpoles sampled
from high-altitude sites differ physiologically from those
from low-altitude sites, in terms of routine metabolic
rate and freeze tolerance?
Results
Adult spawning behaviour in relation to altitude
Temperature on the day of egg mass observation was,
on average, 7.5 ± 2.1°C (Table 1) and did not vary predictably with altitude (r2 = −0.03, p = 0.41). Likewise, no significant regression with altitude was found for the average
temperature in the week prior to egg mass observation
(mean temperature = 4.8 ± 0.9°C; r2 = −0.06, p = 0.49). Degree days prior to egg mass collection was highly variable
across sites (24.5 ± 19.1; Table 1) but also did not show a
significant relationship with altitude (r2 = 0.26, p = 0.09).
The date of egg mass collection was on average 30 days
later at high- compared to low-altitude sites (Table 1)
and Julian spawning day showed a significant positive
Table 1 Spawning date and temperature by mountain and altitude, shown as the date of egg mass observation
(observation date) and corresponding Julian day; alongside the degree days prior to egg mass observation, the daily
mean temperature on the day of egg mass observation (observation day temp; °C), and the mean temperature of the
week prior to egg mass observation (week prior temp; °C)
Mountain
Altitude
Observation date
Julian day
Degree days
Observation day temp
Week prior temp
DUB
DUB
HIGH
19-Apr
109
30.6
7.1 ± 7.1
4.7 ± 5.4
LOW
23-Mar
82
33.5
6.8 ± 4.6
5.6 ± 5.5
IME
IME
HIGH
02-Apr
92
NA*
NA*
NA*
LOW
24-Feb
55
1.5
5.8 ± 0.3
4.3 ± 0.9
LAW
LAW
HIGH
15-Apr
105
31.1
5.5 ± 2.1
4.3 ± 4.2
LOW
21-Mar
80
22.8
8.3 ± 2.6
3.3 ± 4.0
LOM
LOM
HIGH
09-Apr
99
62.9
10.8 ± 4.5
6.0 ± 3.9
LOW
01-Mar
60
5.5
4.5 ± 0.6
5.3 ± 1.1
MNT
MNT
HIGH
10-Apr
100
28.0
9.8 ± 4.0
5.9 ± 4.1
LOW
21-Mar
80
5.1
8.8 ± 2.4
4.2 ± 4.6
Observation day temp and week prior temp are mean values and are accompanied by their standard deviation.
*Data not available due to logger failure.
Muir et al. BMC Evolutionary Biology 2014, 14:110
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80
60
70
Julian day
90
100
110
Between eight and 20 individuals per site were measured for RMR, due to varying levels of mortality (Mean =
16 ± 5; Table 2). Mean RMR per site varied between
0.02 ml O2 g−1 h−1 (LOMHIGH) and 0.10 ml O2 g−1 h−1
(DUBLOW), with an overall average of 0.07 ± 0.02 ml
O2 g−1 h−1 (Table 2). Mountain, altitude, and their interaction were found to be significant in predicting RMR.
A Tukey’s HSD test showed a significant difference between high- and low-altitude RMR in individuals from
three of the mountains: DUB (diff = 0.03, p < 0.01), MNT
(diff = 0.02, p = 0.03) and LOM (diff = −0.07, p < 0.01)
(Table 2). The difference between high- and low-altitude
RMR was not significant for IME (diff = −0.01, p = 0.49)
and LAW (diff = 0.01, p = 0.94). The direction of the relationship varied between mountains, with individuals from
DUB, LAW and MNT showing a trend for lower RMR at
high- compared to low-altitude, whereas individuals from
IME and LOM had higher RMR at high-altitude (Figure 3).
The post hoc power analysis of the ANOVA used in the
Tukey’s HSD revealed an effect size of 0.29 giving an
achieved power of 0.69 to determine a significant difference between the means of RMR by altitude within
each mountain. The power to confidently conclude that
200
400
600
800
DUB
IME
LAW
LOM
MNT
0
Larval physiology in relation to altitude
Routine metabolic rate
Average temperature (°C)
relationship with altitude (r2 = 0.80, p < 0.01): individuals spawned 5 days later for every 100 m gain in altitude (Figure 1). The daily mean temperature at all sites
had exceeded the threshold value of 5°C in the week
prior to egg mass collection (Figure 2).
8
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0
Days before spawning
Figure 2 The average daily temperature for the week prior to
egg mass observation (days before spawning) for each site,
seen as a linear regression line of the points. Solid lines show
high- and dashed lines show low-altitude sites per mountain. The
black horizontal line shows the 5°C threshold generally considered
to limit activity in R. temporaria.
no significant interactions have been missed (a type II
error), is generally set at 0.8 [48].
Freeze tolerance
Ten individuals per site were tested for freeze tolerance,
except for LOMHIGH (seven individuals) and DUBLOW
(zero individuals) (Table 2), due to variable tadpole mortality prior to the experiment. Between 10% (LOMLOW,
MNTLOW and LAWLOW) and 80% (IMEHIGH) mortality was observed post-freezing across sites (Mean
survival = 0.60 ± 0.28; Table 2). Out of 50 tadpoles tested
for freeze survival from low-altitude sites, 41 survived
(82%), compared with 12/37 (32%) tadpoles from highaltitude sites (Figure 4). Altitude and weight, but not
their interaction, significantly changed the log likelihood when removed from the GLMM and were thus
included in the final model. There was no significant effect on the log likelihood of the model when mountain
was removed from the model and thus mountain was not
included in the final model (Additional file 1). The results
of the GLMM using the final model (RMR ~ altitude +
weight) showed that individuals from low-altitude sites
had significantly higher survival than those from highaltitude sites (z = 4.20, p < 0.01).
1000
Altitude(m)
Figure 1 The Julian day at which spawn was first observed by
altitude, fitted with the linear regression line: Julian day =
(0.05 × Altitude) + 63.14(r2 = 0.80, p < 0.01).
Discussion
Adult spawning behaviour in relation to altitude
No significant relationship was found between spawning
date and either the temperatures recorded on the day of,
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Table 2 Physiological trait variation by mountain and altitude measured in a common environment
Mountain
Altitude
RMR n
RMR (ml O2 g−1 h−1)
DUB
HIGH
8
0.07 ± 0.02
DUB
LOW
19
0.10 ± 0.02
IME
HIGH
20
0.06 ± 0.02
IME
LOW
19
0.05 ± 0.01
LAW
HIGH
10
0.08 ± 0.02
LAW
LOW
19
0.09 ± 0.02
LOM
HIGH
14
0.09 ± 0.03
LOM
LOW
12
0.02 ± 0.01
MNT
HIGH
20
0.07 ± 0.01
MNT
LOW
20
0.09 ± 0.02
Tukey’s HSD Diff between means
0.03
−0.01
0.01
−0.07
0.02
Tukey’s HSD p value
<0.01*
0.49
0.94
<0.01*
0.03*
Freeze n
Survival
0
NA
10
0.70
10
0.20
10
0.70
10
0.40
10
0.90
7
0.29
10
0.90
10
0.40
10
0.90
Shown are the number of individuals per site measured (n), the mean routine metabolic rate (RMR) and the proportion of survivors following freezing (Survival).
Standard deviations are indicated for RMR, whereas freeze survival is shown as a single measurement per site. The results of the Tukey’s HSD test of significant
difference between the means of RMR of individuals from low- and high-altitude sites, by mountain are shown (Tukey’s HSD p value). A positive difference
between the means (Diff between means) shows that individuals from high-altitude have a lower mean RMR than those from low-altitude, and a negative
difference between the means shows that individuals from low-altitude have a lower RMR than those from high-altitude.
*Significant at p < 0.05.
NA: No freeze tolerance results are available for DUBHIGH due to complete tadpole mortality prior to the freeze tolerance experiment.
0.02
0.04
0.06
0.08
or in the week prior to, egg mass observation that would
have suggested that breeding occurs at lower temperatures at high altitude sites. Degree days also did not
show a significant relationship with altitude but they
were highly variable across sites (Table 1), suggesting
that degree days are not an accurate predictor of spawning activity in R. temporaria. All sites had exceeded the
5°C temperature threshold generally thought to initiate
activity and breeding in R. temporaria [44] in the week
DUB
IME
LAW
LOM
MNT
Low
Altitude
Figure 3 Routine metabolic rate by mountain and altitude. The
mean routine metabolic rate per site is shown by a circle, with the bars
representing the standard deviation around the mean. Low- and
high- altitude sites within each mountain are linked using a straight
line; a positive line shows that RMR is higher at low- vs. high-altitude,
whereas a negative line shows that RMR is lower at high- vs.
low-altitude.
prior to spawning (Figure 2). Our results therefore support
5°C as the activity threshold for R. temporaria regardless
of altitude of breeding site, and demonstrate that
high-altitude individuals experience a longer period of
low-temperatures and delayed spawning compared to
low-altitude individuals. The date of spawning was 5 days
later for every 100 m gain in altitude (Figure 1). As mean
annual temperature decreases by 0.65°C for every 100 m
increase in altitude in this system [40], we can infer that
spawning is on average one day earlier for every 0.65°C
increase in mean annual temperature. However, further
information regarding the date of spawning between
223 m and 720 m (currently not available for this system)
is needed to confirm this pattern. It is interesting to note
that for the mountains with the highest high-altitude sites
(>900 m: DUB, LAW and MNT), the average time difference between spawning at low-and high-altitude is only
24 days, compared with 38 days observed difference between spawning at low- and high-altitude in IME and
LOM (high-altitude sites at 703 m and 720 m, respectively;
Tables 1 and 3). This could suggest that high-altitude
individuals at the highest breeding sites are breeding at
a lower temperature. However, this is not reflected as a
significant difference between high- and low-altitude
spawning temperatures in these mountains (Table 1). The
longer period of low temperatures at high- compared to
low-altitudes prior to spawning supports the hypothesis
that high-altitude individuals experience a shorter annual
activity period but does not support the hypothesis that
breeding occurs at lower temperatures at high altitudes to
provide a longer developmental period, facilitating survival
at high-altitude within R. temporaria.
Breeding at a lower temperature would allow individuals to spawn earlier in the year, thus providing their
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0.0
0.2
Mortality
0.4
Freeze survival
0.6
0.8
Survival
1.0
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High
Low
Altitude
Figure 4 The proportion of individuals from high- versus low-altitude sites that survived freezing.
offspring with a longer period in which to develop prior
to metamorphosis [20], such as has been found for amphibian species at higher latitudes compared to their
lower latitude counterparts [20] but, to the best of our
knowledge, has not yet been observed within-species.
However, phenological studies that have quantified withinspecies breeding temperature have typically used local
weather station data [49,50] and so the spatial scale might
not be not fine enough to represent local conditions in
mountain areas, where temperatures can vary rapidly over
short geographical distances [21,47,51]. Therefore, such
studies are liable to miss within-species differences in
spawning temperature in relation to altitude. When we
quantified temperature at a local level, our results support
the absence of a shift in breeding temperature in lowtemperature environments, contributing to knowledge of
the fundamental niche of R. temporaria. However, we
did find that R. temporaria can substantially alter the
date they breed in response to the environmental conditions experienced, even between geographically close
breeding sites, demonstrating a high degree of plasticity
in terms of breeding timing.
Larval physiology in relation to altitude
Routine metabolic rate
There was a significant interaction between mountain and
altitude, which complicated interpretation of RMR. Posthoc tests indicated no significant differences in RMR
Table 3 Locations of study sites in Scotland including site name (study mountain and whether high- or low-altitude)
with associated abbreviation, latitude, longitude and altitude (metres above sea level)
Site
Abbreviation
Latitude
Longitude
Beinn Dubhchraig High
DUBHIGH
56.3951
−4.7506
Altitude
900
Beinn Dubhchraig Low
DUBLOW
56.4212
−4.6945
197
Beinn Ime High
IMEHIGH
56.2347
−4.8123
703
Beinn Ime Low
IMELOW
56.2046
−4.7628
155
Ben Lawers High
LAWHIGH
56.5423
−4.2291
990
Ben Lawers Low
LAWLOW
56.5002
−4.2354
215
Ben Lomond High
LOMHIGH
56.1857
−4.6478
720
Ben Lomond Low
LOMLOW
56.1598
−4.6363
77
Meall nan Tarmachan High
MNTHIGH
56.5188
−4.2958
900
Meall nan Tarmachan Low
MNTLOW
56.4994
−4.2523
223
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between tadpoles sampled from high and low sites on
IME or LAW. In regards to the other three mountains,
there were significant differences but not always in the
same direction: for DUB and MNT tadpoles sampled from
high elevation showed a decreased RMR relative to those
from low elevation, but the largest difference due to elevation was at LOM, where tadpoles from high elevation
showed a substantial increase in RMR compared to those
from low elevation (Figure 3; Table 2). However, due to
the moderate power of the model (power = 0.69), it is possible that some significant relationships were missed. It
has been suggested that a lower resting metabolic rate can
allow more energy to be allocated to growth in resourcelimited environments [16] and a link between lower RMR
and increased growth rate has been found in the southern
toad [29], Sydney rock oyster [30] and snapping turtle
[31]. Furthermore, Sears [6] found that both increased
growth rates and reduced RMR were positively correlated
with altitude in sagebrush lizards. The three mountains in
our study system where RMR was lower in individuals
from high-altitudes (although the difference was very
small for individuals from LAW) have been shown to be
locally adapted to temperature parameters, with larval
period decreasing and growth rate increasing at highaltitude [40]. Therefore, the lower RMR of individuals
from high-altitude from DUB, MNT and LAW, is in line
with the increased growth rates observed at these sites.
Lindgren and Laurila [16] did not find a link between
growth rates and RMR in R. temporaria along a latitudinal
gradient in Sweden. Therefore, this is the first tentative
evidence of reduced RMR being linked to increased
growth rate as an adaptation to low-temperatures in an
amphibian. However, the significantly higher RMR at
high- than low-altitude in LOM (Figure 3), and the nonsignificant difference between LAWHIGH and LAWLOW
in terms of RMR, could suggest that local conditions other
than altitude are also important in driving divergence in
RMR. For instance, the lowest RMR were observed in individuals from IMELOW and LOMLOW, where spawning
occurred at the lowest temperatures of all the sites (apart
from LAWHIGH), and this could suggest that low RMR is
beneficial in tolerating low temperatures at all altitudes,
particularly early in the season when temperatures can
fall after spawning. It has also been speculated that increased growth rate can be a result of increased time
spent foraging, facilitated by lower predator presence in
low-temperature environments, and is unrelated to RMR
[52-54]. Furthermore, RMR is influenced by temperature
and there was up to a 3°C difference in temperature whilst
measuring RMR of individuals from different sites, as well
as a difference between the temperature at which the tadpoles were raised (15°C) and the temperature at which
RMR was measured (19-22°C). Therefore, further research
is needed to assess whether site-specific differences in
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RMR remain constant through time and whether other
mountains with breeding sites of above 900 m also show
reduced metabolic rate at high- vs. low-altitude.
Freeze tolerance
Just over half of the tadpoles that were frozen survived
(61%; Table 2) and altitude was significant in predicting
freeze survival: individuals sampled from low-altitude
survived freezing significantly better than those from
high-altitude (z = 4.20, p < 0.01). Voituron et al. [3] suggested that R. temporaria adults were freeze intolerant, as
100% mortality was observed after eight hours of complete
bodily fluid freezing. However, Pasanen and Karhapää [55]
found that R. temporaria adults could survive 24 hours
in a sub-zero environment but died within three days
(the actual period an individual was frozen was not
measured in that study). Our results suggest that tadpoles of R. temporaria are also capable of surviving
short periods of freezing. This is the first time, to the
best of our knowledge, that larval freeze tolerance has
been demonstrated in any amphibian.
Although the results presented here suggest that tadpoles are capable of surviving being frozen, the finding
of a greater survival of low- compared to high-altitude
individuals appears counterintuitive, given the longer
period of sub-zero temperatures at high- than lowaltitude in Scotland [33] and the sub-zero average winter temperature of −2.2°C at high-altitude, compared to
1.0°C at low-altitude at the breeding sites used in this
study [45]. Indeed an increase in freeze tolerance with
altitude has been found in plants (Arabidopsis thaliana
[56]) and insects [18,57], but freeze tolerance and altitude
has not been explicitly linked in herptiles. However,
evolution of freeze tolerance in frogs, lizards and turtles
has been linked to ecological pressures relating to winter temperatures experienced [35,58,59]. In general, freeze
tolerant species are those that terrestrially overwinter in
sub-zero temperatures, as opposed to avoiding freezing in
deep water bodies [60]. Therefore, a higher freeze tolerance would suggest that low-altitude tadpoles are more
often exposed to freezing temperatures, whereas highaltitude tadpoles may avoid such exposure altogether by
inhabiting deep water bodies or metamorphosing prior to
winter. Formation of deep water pools is inhibited at highaltitudes in Scotland due to the rocky, exposed landscape
[61]. Therefore, freeze exposure is more likely avoided in
high-altitude tadpoles by metamorphosing within a single
active season, facilitated by a faster growth rate in conjunction with a lower RMR ([45], this study). Therefore,
our results potentially suggest that of the tadpoles found
overwintering in Scotland [19], low-altitude individuals
are more likely to overwinter as tadpoles than highaltitude individuals. However, it is possible that long periods of snow cover at high-altitude [33] could insulate
Muir et al. BMC Evolutionary Biology 2014, 14:110
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tadpoles from freezing temperatures even in shallow water
bodies. Therefore, further field research is needed to assess
whether fewer, if any, high- than low-altitude individuals
overwinter as tadpoles.
Conclusion
R. temporaria adults do not show behavioural adaptations in terms of breeding at lower temperatures, instead
they delay spawning based on the temperature experienced. However, R. temporaria larvae appear to have the
potential to physiologically adapt to low-temperature environments, although these relationships, and the results
we obtained for routine metabolic rate and freeze tolerance, are not always straightforward to interpret. Therefore, our results suggest that survival at high-altitude
may be facilitated by physiological mechanisms that permit faster growth rates, allowing completion of larval development within a shorter period of time, alleviating
the need for adaptations that extend the time available
for larval growth. How individuals respond to environmental temperature at a local level is an important step
in relating ecological and evolutionary pressures to
phenotypes.
Methods
Adult spawning behaviour in relation to altitude
Data collection
Paired high- (above 700 m; R. temporaria occur to over
1000 m in Scotland [43]) and low-altitude (below 300 m)
sites from five mountains within west central Scotland
were selected for study (Table 3). The study sites have low
neutral genetic population structuring (high between-site
gene flow) and show local adaptation of larval traits in
relation to temperature [40,45]. Air temperatures were
recorded every two hours at each site between March
2010 and October 2011 using Thermocron i-buttons
(Dallas Semiconductor/Maxim, London) and downloaded
to a laptop every six months using a USB i-button adapter
(Dallas Semiconductor/Maxim, London) and the software,
Thermodata viewer (Thermodata pty Ltd., Melbourne)
(as per [40,45]). Sites were visited from early February
2011 and date of spawning was recorded as the day egg
masses were first observed at each site. The daily mean
temperature on the day of egg mass observation was
calculated for all sites. Although the majority of egg
masses were at or below Gosner stage 10 on this date,
and thus likely to have been laid no more than 100 hours
previously [62], it is possible that spawning activity
started prior to this. Therefore, the daily mean temperatures and the overall average for the week prior to egg
mass observation were also calculated. In addition, degree days to egg observation were calculated for each
site (a statistic commonly used to predict flowering date
in plants; [63]), using the following approach: From the
Page 8 of 11
1st January 2011, degrees above the threshold for development (set at 5°C) were calculated per day using the
formula: ((daily maximum temperature + daily minimum
temperature)/2)-threshold value. The resulting values were
summed to give the total degree days [63].
Statistical analyses
Linear regression models were used to assess whether
the temperature and date at which individuals spawned
had a significant relationship with altitude (continuous
variable; m) using: 1) the date parameter, Julian day; and
2) the temperature parameters: mean temperature on
the date spawn were observed, the mean temperature
for the week prior to spawn observation, and degree
days over the threshold 5°C.
Larval physiology in relation to altitude
Sampling
Ten R. temporaria egg masses were collected soon after
laying (Gosner stage 10 or below; [64]) from each of the
sites monitored for adult breeding phenology. Egg masses
were collected during the 2011 breeding season, transferred to the laboratory and maintained in individual sterilised water tanks until hatching at 10°C. At hatching
(Gosner stage 22), a randomly selected subset of ten tadpoles were removed from each egg mass and placed in
groups of five in two individual 1.3 L plastic baskets with a
0.1 cm mesh. Baskets were placed in large tanks in a common 15°C treatment room. Water quality was maintained
using a flow-through system and tadpoles were fed ad
libitum with a 1:2 mixture of finely ground dried fish
and rabbit food (for further details see [45]).
Routine metabolic rate
Tadpoles were allowed to develop until hind leg toe differentiation became apparent, in the early stages of metamorphosis (Gosner stages 36–39). Twenty individuals per site
(one individual per basket*two baskets per family*ten families per site) were transferred to individual containers and
allowed to acclimatise to laboratory conditions in 100%
oxygenated water for an hour prior to commencement of
experimental procedures. After this period, tadpoles were
moved into 8 ml respiration tubes filled with 100% oxygenated water and the lids immediately sealed. Tubes were
placed in a dim, quiet location to reduce disturbance during the experiment. Respiration tubes remained closed for
one hour. At the end of this period, the lid was removed
and the oxygen saturation of the water was measured
using an oxygen meter and probe (Strathkelvin Instruments, UK). The same procedure was carried out using a
control tube containing no tadpole, to account for any
oxygen consumption caused by microbial action. The oxygen meter was calibrated prior to each use using 100%
and 0% oxygenated distilled water as standards. Distilled
Muir et al. BMC Evolutionary Biology 2014, 14:110
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water was fully oxygenated using an aquatic bubbler and
fully deoxygenated by adding sodium sulphite [65]. The
oxygen probe was maintained at a constant temperature
to avoid biases caused by thermal fluctuation using a flowing water bath. The temperature of the water bath was
monitored throughout using a submerged thermometer.
Once the experiment was completed, tadpoles were immediately blotted dry to remove excess water, weighed
using an electric balance (to the nearest 0.1 g) to account
for size differences among individuals in metabolic rate
calculations, and returned to 100% oxygenated water. Although tadpole activity levels were very low whilst sealed
in the respiration tubes, some short bursts of spontaneous
activity were observed. Therefore, the metabolic rate estimates are considered as routine, which includes resting
metabolic rate plus any extra energy expenditure due to
spontaneous activity and stress [16]. Percentage of oxygen
consumed was calculated by subtracting the oxygen saturation of the control tube (i.e. the oxygen used by microbial
activity) from the oxygen saturation of each respiration
tube (i.e. the total oxygen consumed by both tadpole and
bacterial activity). Percentage saturation was converted to
ml l−1 using standard conversion tables based on water
temperature during the experiment (water temperature
varied between 19-22°C depending on the date of the experiment). Routine metabolic rate (RMR) was calculated
for each individual as millilitres of oxygen consumed per
gram weight per hour (ml O2 g−1 h−1).
Freeze tolerance
Ten individuals per site (one individual per family*ten
families per site; different individuals to those used in the
RMR experiment) were moved to individual containers at
Gosner stage 36–39 (hind leg toe differentiation) and deprived of food for 48 hours. Tadpoles were sealed within
individual containers containing 80 ml of water and
cooled to 4°C for 24 hours to cause inactivity. Containers
were then gradually cooled, over a period of six hours, just
until all the water in the container became completely
frozen. Following this, tanks were gradually warmed
(over a period of 14 hours) to 15°C and this temperature
was maintained for one hour. All tadpoles were assessed
for normal swimming behaviour at this point and the
number of individuals that were still alive and exhibiting
normal behaviour were recorded as the measure of
freeze survival.
Statistical analyses
To evaluate whether RMR or freeze survival varied by
altitude (fixed factor of interest; considered as a categorical variable of low or high) a generalised linear mixed
model approach (GLMM) was used, as implemented in
R v2.12.1 [66] using the lme4 package [67]. Mountain
was included as a random factor in all models and family
Page 9 of 11
was nested within altitude in the RMR model, but multiple individuals from the same egg mass were not used
in the freeze tolerance experiments thus family was not
included in the freeze tolerance model. Weight was also
included as a linear covariate in the freeze survival
models, but was already accounted for in the measurement of RMR (ml O2 g−1 h−1). For RMR the model was
assessed under a normal distribution and for freeze survival a binomial distribution was used. Each model parameter and interaction was sequentially removed from
the models and a likelihood ratio test used to evaluate
parameter significance. Only parameters that significantly changed the log likelihood when removed from
the model were included in the final model. Significant
differences due to the model parameters that showed
interactions were evaluated using a Tukey’s HSD test [68],
which works in conjunction with an ANOVA of the
GLMM. A post-hoc power analysis of the ANOVA was
carried out for RMR using G*Power v3.1.3 [69]; effect
size was calculated using the formula in Krebs [70].
Animal ethics statement
All the protocols used in this study were approved by
the U.K. Home Office: Project License 60/4110.
Availability of supporting data
The data sets supporting the results of this article are
available in the Dryad repository, doi:10.5061/dryad.ks2j1,
https://datadryad.org/resource/doi:10.5061/dryad.ks2jl [71].
Additional file
Additional file 1: The results of the likelihood ratio test for the
freeze tolerance data. Removal of mountain from the model did not
significantly change the log likelihood (Models 4 and 8) and reducing the
complexity of the model from Altitude*Weight to Altitude + Weight did
not significantly affect the log likelihood (Models 5 and 8). Therefore,
Model 8 was chosen as the final model and used to run the GLMM.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
This research forms part of APM’s PhD thesis work on the population
genetics of R. temporaria in Scotland with BKM and RB; she was responsible
for all aspects of the work, from experimental design, sampling, experimental
procedures, analyses and writing. BKM contributed to experimental design,
advice on analyses, and editorial content. RB contributed to editorial content.
All authors read and approved the final manuscript.
Acknowledgements
We thank Rose Hanley-Nickolls, Romaine Furmston-Evans, David Fettes and
Martin Muir for assistance with fieldwork; and Aileen Adam and Elizabeth
Kilbride for laboratory support. Thanks to the landowners that permitted
access to sites. Fieldwork was supported by grants from the Royal
Geographic Society, the Glasgow Natural History Society and the Scottish
Mountaineering Trust. Permission for sampling from protected areas was
granted by Scottish Natural Heritage. This study was supported by PhD CASE
studentship funding from the Biotechnology and Biological Sciences
Muir et al. BMC Evolutionary Biology 2014, 14:110
http://www.biomedcentral.com/1471-2148/14/110
Research Council, in partnership with the Royal Zoological Society of
Scotland.
Received: 17 February 2014 Accepted: 14 May 2014
Published: 23 May 2014
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doi:10.1186/1471-2148-14-110
Cite this article as: Muir et al.: Behavioural and physiological
adaptations to low-temperature environments in the common frog,
Rana temporaria. BMC Evolutionary Biology 2014 14:110.
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